11: GPC

Transcript 11: GPC

ADVANCED BIO-FRIENDLY POLYMERS Gel permeation chromatography – a tool for determination of molar mass of polymers

Igor Lacík

Molecular weight: characterization techniques

n    techniques related to colligative properties (dependence on the number of molecules)  membrane osmometry (> 25 000 g/mol)  vapor pressure osmometry (< 25 000 g/mol) mass spectrometry  electrospray ionization mass spectrometry (ESI-MS)  matrix-assisted laser desorption/ionization (MALDI) size exclusion chromatography

w    static laser light scattering analytical ultracentrifuge size exclusion chromatography

z    static laser light scattering analytical ultracentrifuge size exclusion chromatography

   viscometry size exclusion chromatography

Molecular weight: characterization techniques

w    static laser light scattering analytical ultracentrifuge size exclusion chromatography

z    static laser light scattering analytical ultracentrifuge size exclusion chromatography

   viscometry size exclusion chromatography

Size-exclusion chromatography – principle

1. Column separation technique 2. Based on

enthalpy-free partitioning

of analyzed solutes, most often the polymer chains, of different molecular weight / length (size) between mobile and stationary phases

Separation mechanism is based on the size

• • •

Stationary phase pore size pore size distribution particle size

•

Mobile phase

 

flow rate in ml/min providing “elution volume, V e ” or elution time, t e ”

Separation mechanism is based on the size

Time 3 Time 2 Time 1

Time 1 < Time 2 < Time 3

e,1

< V

e,2

< V

e,3

Size-exclusion chromatography – principle

1. Column separation technique 2. Based on

enthalpy-free partitioning

of analyzed solutes, most often the polymer chains, of different molecular weight / length (size) between mobile and stationary phases

V e



0 

K d

(

V t



0 ) K d - partition coefficient 0  K d  1 solute exluded solute permeated V e - elution volume for given size V 0 - interstitial volume (between particles of column packing) V t - total available volume of column (interstitial and pore volumes)

Size-exclusion chromatography – setup

Injection of polymer

of unknown molecular weight characteristics

Pump

eluent

Commercial columns of defined characteristics for column packing:

diameter, exclusion limit, pore-size distribution

Detector signal = f(elution volume)

calibration curve with standards:

elution volume = f(MW)

molecular weight characteristics (MWD, M w , M n ) of polymer molecular weight distribution

exclusion separation permeation log M

GPC signal



(log

)  ln( 10 )

M f w

(

)  ln( 10 )

f n

(

)

Size-exclusion chromatography – hardware

PUMP INJECTOR COLUMNS MALLS DEGASSER RI SOFTWARE MAREK Degasser Rheodyne inj . or Waters Autosampler Pump Waters 515 Columns MALLS SLD7000 DRI Detector Waters 2410 WinGPC®7.4 Software

Size-exclusion chromatography – calibration SEC is an indirect method

 needs RELATION of elution volume (volume slice) to molecular weight = calibration • several types of calibration 1. narrow distributed standards of the same polymer

M w /

[  ] 1

1  = [ 1.1

 ] 2 M 2 ~ size of coil ~ V elution (Benoit et al, 1966) note: a) Mark-Houwink equations is related to MW range b) careful in low MW range where a ~ 0.5 J.Polym.Sci. 6, 1759 (1968) 3. absolute molecular weight detectors (LS, viscosity) 4. effective calibration (no match between standards and analyzed polymer) decrease in accuracy

Size-exclusion chromatography – hints/experience

Remember  suppress interactions to avoid non-SEC phenomena

(1)

polymer - polymer (aggregation) – earlier elution, artificially increased molecular weight

(2)

polymer - column packing (adsorption) - delayed elution, artificially decreased molecular weight

(3)

eluent-column packing (repulsion) – earlier elution, artificially increased molecular weight

(4)

polymer - eluent (aggregation) - earlier elution, artificially increased molecular weight

(5)

etc.

Size-exclusion chromatography – hints/experience

Remember  suppress interactions to avoid non-SEC phenomena 

water-soluble polymers (polyelectrolytes)

Problem •H-bonding •hydrophobic interactions •polyelectrolyte effect •adsorption (ion inclusion) •repulsion (ion exclusion) •calibration •concentration effect •resolution (eluent viscosity) Cure Eluent composition & column selection PSS TosoH Biosep Polym. Laboratories Waters Suprema HEMA GRAM (DMAc) TSK PWXL Ultrahydrogel Direct calibration with standards of well-defined M p Optimized solute concentration Temperature of analysis

Size-exclusion chromatography – hints/experience

Remember  suppress interactions to avoid non-SEC phenomena 

water-soluble polymers (polyelectrolytes like poly (acrylic acid))

       eluent: 0.1M LiNO columns: 3 , 0.04 M phosphate bufer, pH 8.0, 0.01M NaN Suprema 100, 1000, 3000, 10 00 data acquisition and evaluation 0 Å (8 x 300 mm, 10 3 m m) flow rate 0.6 -1.0 ml/min (ethylene glycol – internal standard) temperature 60-80ºC calibration p(NaA) standards 1 250 – 1 100 00 0 g/mol (PSS, Mainz, Germany) sample: concentration 0.5 - 2 mg/ml, injection volume 200 m L WinGPC 7 (PSS, Mainz, Germany) or 0.1M Na 2 HPO 4 (pH 9) or other compositions EG system peaks - salt excluded from PE region sample

Size-exclusion chromatography – hints/experience

Remember  suppress interactions to avoid non-SEC phenomena Calibration curve (polyacrylic acid) Guide-lines: • no upward curvature at high M.w, i.e. no adsorption of PAA to column packing (solute-gel interactions) pronounced at  M.w.

• variation of ionic strength - test to estimate the interactions  ionic strength too low: elution volumes  increase due to electrostatic interactions ionic strength too high: elution volumes  increase due to hydrophobic interactions remove charged monomer by dialysis: as it adds to the ionic strength of eluent 

a suitable ionic strength is in case of the lowest elution volumes

Size-exclusion chromatography – hints/experience

Remember  suppress interactions to avoid non-SEC phenomena 0.4

0.3

0.2

0.1

0.0

0.8

0.7

0.6

0.5

1.1

1.0

0.9

PAA concentration in mg/ml Concentration effect (polyacrylic acid) 5.0

22.0

8E+5 6E+5 3.0

21.0

4E+5 1.0

0.7

0.3

10 3 10 4 Mol mass 10 5 10 6 g/mol 20.0

0 1 2 3 4 5  decrease in hydrodynamic volume of polymer coil at higher polymer concentration leads to the artificially lower M.w.

 lowering the sample concentration until the constant molar mass is reached yet detector sensitivity is sufficient 6 2E+5

Size-exclusion chromatography – hints/experience

Accuracy of the flow rate (polyacrylic acid) ! EXTREME SENSITIVITY TO VARIATION IN THE FLOW RATE ! Elution volume of internal standard [ml] Difference [%] M p [g/mol] Difference in M p [%] sample EG

36,525

36,342 36,160 35,794 36,707 36,890 37,256

- 0,5 - 1,0 - 2,0 + 0,5 + 1,0 + 2,0

57 700

55 000 50 700 42 600 64 300 69 300 80 000

V adjusted sample



V analysis sample V calibratio

int .

tan

n dard V analysis

int .

tan

dard

 it is essential flow rate is controlled by the flowmarker (ethylene glycol) which also controls the pore volume and any changes on columns

- 4,7 -12,1 -26,2 11,4 20,1 38,6

Size-exclusion chromatography – hints/experience

Effect of temperature of analysis (polyacrylic acid) 2.0

1.5

1.0

0.5

room T Water Toluene THF 0.0

0 20 40

Temperature [ C]

60 80   the partitioning of the solute between mobile and stationary phases depends on viscosity of mobile phase: improved partitioning  improved resolution viscosity of aqueous mobile phase can be adjusted to that of the organic one by the temperature of analysis

Size-exclusion chromatography – hints/experience

Final hints with special emphasis to the aqueous-phase SEC • fast advancing in the last years with strong achievements (columns and new experience) • there exist more and more examples of very precise analysis with high resolution (a strong improvement in aqueous-phase SEC) • patience in thorough testing after thinking of all the possibilities which may hamper the experiment • column history may play a role (more in aqueous-phase SEC than in aqueous-phase SEC)

Size-exclusion chromatography – final slide

This is also the common SEC analysis

